A community cluster of influenza A(H3N2) virus infection with reduced susceptibility to baloxavir due to a PA E199G substitution in Japan, February to March 2023

A community cluster of influenza A(H3N2) caused by viruses with an E199G substitution in PA was detected in Nara, Japan, between February and March 2023. The three patients with these mutant viruses had not received antiviral treatment before specimen collection but patients in the same hospital had. The sequences of the mutant viruses were closely related, suggesting clonal spread in Nara. They showed reduced susceptibility to baloxavir in vitro; however, the clinical significance of the PA E199G substitution remains unclear.

A community cluster of influenza A(H3N2) caused by viruses with an E199G substitution in PA was detected in Nara, Japan, between February and March 2023.The three patients with these mutant viruses had not received antiviral treatment before specimen collection but patients in the same hospital had.The sequences of the mutant viruses were closely related, suggesting clonal spread in Nara.They showed reduced susceptibility to baloxavir in vitro; however, the clinical significance of the PA E199G substitution remains unclear.
Baloxavir marboxil, a cap-dependent endonuclease inhibitor, was approved in Japan in 2018 for the treatment and prophylaxis of influenza A and B virus infections in patients 12 years and older and children younger than 12 years weighing at least 10 kg.Since the 2017/18 influenza season, we have been monitoring the susceptibility of circulating influenza viruses to baloxavir and the neuraminidase (NA) inhibitors oseltamivir, peramivir, zanamivir, and laninamivir.Approximately 10-15% of total isolates are randomly selected and analysed.During our nationwide monitoring, we detected sporadic cases of influenza A(H1N1) pdm09 and A(H3N2) viruses with reduced susceptibility to baloxavir in the 2018/19 and 2019/20 seasons [1][2][3].These viruses were detected in children younger than 10 years and possessed E23K or I38T amino acid substitutions in their polymerase acidic subunit (PA).No community clusters of such mutant viruses have been identified to date.
Here, we report a community cluster of influenza A(H3N2) virus with reduced susceptibility to baloxavir due to a PA E199G substitution.

Detection of PA E199G mutant influenza A(H3N2) viruses
In Japan, influenza activity was low throughout the COVID-19 pandemic [4]; however, during the 2022/23 season, Japan experienced its first influenza outbreak since the 2019/20 season.Baloxavir was the most used influenza antiviral during the 2018/19 season in Japan with ca 5.3 million doses [5], but the use of baloxavir has since declined due to the detection of resistant viruses and low influenza activity.The doses of baloxavir supplied to medical institutes during the 2019/20, 2020/21, 2021/22 and 2022/23 season were 600,000, 97,000, 12,000 and 590,000, respectively [16].Influenza A(H3N2) viruses predominated, followed by A(H1N1)pdm09 and B/Victoria-lineage viruses.
Deep sequencing analysis revealed that the PA E199G substitution was present in the specimens.No amino acid substitutions associated with reduced susceptibility to NA inhibitors were detected in the PA E199G mutant viruses.

Frequency of the PA E199G substitution in influenza A(H3N2) viruses
To assess the frequency of the PA E199G substitution in A(H3N2) viruses, we examined 81,806 PA sequences from influenza A(H3N2) viruses between 1968 and August 2023 deposited in the GISAID EpiFlu database (https://gisaid.org)(Table 2).The PA 199E position is highly conserved and only nine (0.01%) PA E199G mutant viruses were identified worldwide: one in the 2014/15, one in the 2015/16, two in the 2018/19, four in the 2021/22 and one in the 2022/23 season (excluding the ones from Nara).For those seasons, the number of PA sequences available for this analysis was 4,218, 2,809, 9,293, 20,230 and 17,304 in the 2014/15, 2015/16, 2018/19, 2021/22 and 2022/23 seasons, respectively.The frequency of the PA E199G substitution was therefore 0.02%, 0.04%, 0.02%, 0.02% and 0.006% in these seasons, respectively.Two PA E199G mutant viruses (A/Catalonia/NSVH554225590/2022 and A/Catalonia/NSVH554225591/2022) belonging to clade 2a.1 were detected on 19 April 2022 in Catalonia, Spain.At least 96 influenza A(H3N2) viruses isolated in Catalonia in April 2022 were deposited in the GISAID EpiFlu database; among those were no other PA E199G mutant viruses.These results suggest that the frequency of the PA E199G mutant A(H3N2) viruses is low.

A community cluster of the PA E199G mutant influenza A(H3N2) viruses
Although the frequency of the PA E199G mutant A(H3N2) viruses is low, we found that three of 12 influenza A(H3N2) viruses isolated in Nara possessed the PA E199G substitution.No epidemiological link among the patients infected with the PA E199G mutant viruses could be identified, and none of them had received baloxavir before specimen collection.The whole genome sequences of these mutant viruses were identical except for an R723L substitution in polymerase basic protein 1 (PB1) of A/Nara/12/2023, suggesting clonal spread in Nara.These results indicate a community cluster of the PA E199G mutant A(H3N2) viruses in Nara.

Antiviral susceptibility of the PA E199G mutant influenza A(H3N2) viruses
We determined the susceptibilities of the three PA E199G mutant A(H3N2) viruses to baloxavir and NA inhibitors (oseltamivir, peramivir, zanamivir and laninamivir) by using a focus reduction assay and a fluorescence-based NA inhibition assay, respectively, as previously described [8].Our results are expressed as 50% inhibitory concentration (IC 50 ) values.To interpret the antiviral susceptibility, we applied the World Health Organization criteria, which are based on foldchanges in IC 50 values compared with the median value for viruses from the same type/subtype/lineage [5].For NA inhibitors, normal (< 10-fold increase), reduced (10-100-fold increase) or highly reduced (> 100-fold increase) inhibition of influenza A viruses is defined [5].For baloxavir, the provisional criteria define influenza virus inhibition as normal (< 3-fold increase) or reduced (≥ 3-fold increase).

Discussion
The PA E199G mutant influenza A(H3N2) virus was previously detected during treatment-emergent variant monitoring in a paediatric study of baloxavir and showed reduced susceptibility to baloxavir [6].In the study presented here, three PA E199G mutant A(H3N2) viruses (A/Nara/10/2023, A/Nara/12/2023 and A/Nara/14/2023) belonging to clade 2a.3a were detected between 20 February and 3 March 2023 in three patients without prior baloxavir treatment.The primary case of these mutant virus infections could not be identified.However, two wild-type influenza A(H3N2) viruses (A/Nara/3/2023 and A/Nara/8/2023) belonging to the same clade were detected between 30 January and 13 February 2023 in patients who received baloxavir on the day of specimen collection in the same hospital as the patients with the PA E199G mutant virus (Table 1).The whole genome sequences of A/Nara/3/2023, isolated from a teenage child, and the three PA E199G mutant viruses were almost identical except for HA I156M and NA S335G substitutions of A/Nara/3/2023.However, A/Nara/8/2023, isolated from an adult patient, possessed six substitutions compared with the PA E199G mutant viruses: two in polymerase basic protein 2 (PB2), one in PB1, two in PA and one in HA.
An influenza outbreak occurred in the school attended by the patient infected with A/Nara/3/2023.We previously reported that influenza viruses with reduced susceptibility to baloxavir were detected 3-6 days after baloxavir administration [2].These results suggest the possibility that the PA E199G mutant A(H3N2) virus emerged due to the selective pressure of baloxavir and started to spread earlier in the season.In addition, A/ Nara/10/2023 and A/Nara/12/2023 belonged to family clusters where the transmission chain started with children, and A/Nara/14/2023 was detected in a sporadic case.As specimens from other outbreaks were unavailable, we cannot rule out that the PA E199G mutant virus circulated among children.
Of the substitutions that emerged after baloxavir treatment in the past, PA I38 substitutions were the most frequent and had the greatest impact on baloxavir susceptibility [9].The frequency of emergence of PA I38 mutant viruses in children younger than 12 years is higher than that in patients aged 12-64 years [2,6,10].
We previously reported human-to-human transmission of PA I38T and PA E23K mutant viruses in children younger than 10 years [1-3], but no community clusters of these viruses have been identified.The higher frequency of antiviral-resistant influenza viruses in children appears to be associated with longer periods of viral shedding and higher viral titres than in adults [11].In our current study, however, a community cluster of the PA E199G mutant virus was detected among patients in their 30s, 50s and 60s.Further studies are required to evaluate the replication and transmission fitness of the PA E199G mutant viruses.
Although patients infected with the PA I38 mutant viruses exhibited prolonged virus shedding, a rebound in virus titres after treatment and delayed symptom alleviation [10,[12][13][14][15], the significance of the PA E199G substitution and the effectiveness of baloxavir in patients infected with the PA E199G mutant viruses have not been established.Our data suggest that the PA E199G mutant viruses have retained viral fitness and have the potential for human-to-human transmission.Furthermore, accumulation of multiple antiviral-resistant substitutions has a synergistic effect, resulting in a further reduction in the susceptibility to inhibitors [8].Therefore, monitoring amino acid substitutions is essential to protect public health and support clinical management.

Conclusion
The PA E199G mutant influenza A(H3N2) viruses showed reduced susceptibility to baloxavir in vitro; however, the clinical significance of the PA E199G substitution remains unclear.Therefore, baloxavir may be effective in patients infected with these mutant viruses.

Ethical statement
Ethical approval was not necessary because the study used data obtained in routine surveillance.

Table 3 ,
the PA E199G mutant viruses showed normal inhibition with all four NA inhibitors but

Table 3
Antiviral susceptibility of influenza A(H3N2) viruses detected in Nara, Japan, 2022/23 season (n = 12) Mean IC50 values of triplicate reactions were determined by use of a focus reduction assay.b Mean IC50 values of triplicate reactions were determined by use of a fluorescence-based neuraminidase inhibition assay.c Median IC50 values of influenza A(H3N2) viruses without amino acid substitutions associated with reduced susceptibility to baloxavir isolated in the 2022/23 influenza season in Japan (Baloxavir, n = 267; Neuraminidase inhibitors, n = 278). a